JPH1151941A - Protein measuring dry analytical element and protein measuring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To quickly detect and quantify protein with high sensitivity by using an indicator pigment combined with an acrylic amide polymer and a hydroxy carboxylic acid compound under the existence of molybdic acid ions. SOLUTION: This indicator pigment contains an acrylic amide polymer and a hydroxy carboxylic acid compound under the existence of molybdic acid ions. The indicator pigment is advantageously used for measuring the existence and/or concentration of protein in various aqueous fluids, e.g. biological fluids of humans and animals, outflow sewage of food, industries, and cities, and other fluids tested normally. The protein which can be measured is not limited, and peptides, polypeptides, proteins, and compounds containing or coupled with peptides, polypeptides, and/or proteins can be measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a dry analytical element and a method for quantifying a protein in a fluid sample using the element. More particularly, the invention relates to the use of specific dyes and molybdate ions, polymers containing acrylamide, and hydroxycarboxylic acids in said analytical elements.

[0002]

BACKGROUND OF THE INVENTION Medical and medical research and analytical methods for the rapid and accurate determination of chemical and biological substances present in various fluids such as biological fluids. And diagnostic methods are still required. For example, the presence and amount of proteins must be measured quickly and accurately for effective research, diagnosis and treatment of many human diseases.

[0003] A variety of analytical methods have been developed in the last few decades to detect such substances. These assays are more reliable and in some cases are suitable not only for use in kit form but also for automation. Protein assays have traditionally been performed with the reagents involved in the assay, either in solution or in a test device that has somehow removed fluid. Solution methods are widely accepted in this field and generally require an analytical device that often has a complex capillary to handle and transport the solution. In addition, the analyzer used for such an assay requires both complicated operations and a skilled operator to perform both necessary operations and rigorous washing to avoid contamination between samples. There is.

[0004] An alternative to the solution chemistry method is to use a dry analytical element. Due to the interference from coatings such as binders, surfactants and other reagents necessary to promote or facilitate the deposition of substances, the wetting of the sample and maintain the structural integrity of the element, all It should be understood that solution-based analytical assays are not suitable for use with dry analytical elements. Further dry analysis elements
Compartmentation must be done on site to isolate incompatible elements. This is not the case with solution chemistry, which may employ a separate liquid storage and continuous liquid addition.

[0005] Dry analytical elements and their use have been described in numerous publications, including Pierce.
U.S. Pat. Nos. 4,132,528 and 4,782.
No. 6,605, No. 3,992,158, No. 4,2
No. 58,001; Frickery et al., US Pat. No. 4,670,
381; International Patent Application Publication No. WO 82/2601 (1
Published August 5, 982); European Patent Application No. 051,
183 (published May 12, 1982); and European Patent Application No. 066,648 (December 15, 1982).
Public). The entire contents of these cited references are incorporated herein by reference.

[0006] A useful diagnostic indicator for assessing and monitoring a patient's renal function is the concentration of total protein present in urine. The type and amount of protein present in urine fluctuates,
And it relies on the symptoms of certain diseases that cause kidney failure and block the transfer of protein to urine. Examples of proteins that can be found in urine are not particularly limited,
Albumin, intact immunoglobulins, kappa free chains, lambda free chains, retinol binding protein, α-
There are 1-microglobulin and β-1-microglobulin. These proteins vary widely in amino acid composition and molecular weight. An interesting piece of information for clinicians utilizing the total protein screening assay in urine is the total mass of protein per unit volume of the urine sample. The diagnostic assay, ie, the signal measured, should ideally be independent of the type or type of protein present. For example, 50 mg / dL albumin is 50 mg
/ DL should produce the same signal as any other protein. The normal protein concentration range in human urine is about 5-100 mg / dL.

Various means have been used to determine the total protein concentration in a biological material. In many analytical assays, the light signal is measured, for example, the absorbance in the visible or ultraviolet region of the spectrum, which is proportional to the concentration of the protein in the sample. However, in general, the light signal is a function of the type of protein in the sample, which is undesirable, and is not simply related to the mass of the protein present. These methods are
Usually, it relies on one of the following methods to generate a detectable light signal.

[0008] 1. Complexes of proteins with Cu (II) (the Biuret reaction) result in detectable but weak absorption in the visible region of the spectrum. 2. The absorption in the ultraviolet region of the spectrum by the aromatic amino acids of the protein can be measured. 3. The amount of protein can be measured by derivatizing certain amino acids with a chromophore-containing molecule that can be quantified using intrinsic absorption or fluorescence.

4. The dye can be non-covalently bound to a protein to form a dye-protein complex, and the dye can be used to determine the presence or amount of protein in a sample utilizing the dye that results in perturbation of the dye. Some dyes require the presence of a metal ion, such as molybdenum or tungsten, which results in the formation of a non-covalent complex of the dye, metal ion, and protein, which causes a perturbation in the dye's absorption spectrum. Using this perturbation, the presence or amount of protein in the sample can be measured.

[0010] 5. Competition of the protein with the dye for coordination of Cu (II) results in an absorption signal proportional to the concentration of the protein [Coordination complex of Cu (II) / dye binds to the free dye or Cu (II). It has a different absorption spectrum from uncoordinated dyes]. U.S. Pat. No. 4,132,528 relates to a protein assay based on the biuret reaction. '528
The dry test element of the patent is Cu (I
I) and a biuret reagent of a chelating agent such as CuSO
₄ and tartaric acid or a complex of both, and a pH of about 1
It is composed of a buffer that is higher than 2. Proteins in aqueous fluids such as serum or cerebrospinal fluid (CSF) contain 12
Interacts with the biuret reagent under a pH greater than, causing a reaction between the cupric copper and the protein to give a purple color. Since the color intensity is proportional to the protein content of the serum, the level of the protein can be measured by known colorimetric methods. Unfortunately, the dry slide element of the above patent has a sensitivity lower than the desired sensitivity, ie, about 200 mg /
Protein levels below dL cannot be detected.

US Pat. No. 4,786,605 discloses that by replacing the biuret reagent with a previously prepared coordination complex of Cu (II) pyridylazo dye (or free Cu (II) and free pyridylazo dye). A formulation of analytical elements for protein quantification that is more sensitive than the elements of the '528 patent is described. When aqueous protein is added to the assay element, cupric ions are driven out of the complex by the protein and the absorption curve of the Cu (II) dye complex shifts to that of the uncoordinated dye. Therefore, when protein is added, the absorption peak of the Cu (II) / dye complex decreases in proportion to the amount of protein added, so by properly calibrating with a sample containing a known concentration of protein, The unknown concentration of the protein in the liquid sample can be determined by colorimetry. This patent is
These factors make for a very good dynamic range,
It teaches that it has a sensitivity about 30 times greater than elements based on the biuret reaction.

However, using the method of the '605 patent to quantitate protein in urine samples, consistently, about 10-20% of urine samples had a Coomassie Blue solution assay for protein concentration as a reference standard. It shows an unacceptable random positive bias as compared to the value obtained using the method. That is, the specified concentration of protein in a particular patient's urine sample (about 10-20% of the sample population) according to the method of the '605 patent is greater than the concentration measured using a Coomassie Blue-based assay. This random positive bias is due to the presence of a reducing agent, such as ascorbic acid, in certain urine samples that causes the reduction of both reducible groups of the dye, such as Cu (II) and nitro groups. Was presumed to be the cause. Desirably, the dry analytical element is not affected by the deficiencies of the above assays.

[0013] Solution reactions of certain indicator dyes in the formation of complexes with proteins and metal ions under acidic conditions, which produce a measurable color change, are known.
As mentioned above, analytical assays that work well in solution are:
For the reasons mentioned above, they are not easily adopted for dry analytical elements. It is highly desirable that different proteins react equally with the dye to produce a light signal that is related to the mass of the protein present in the sample and independent of the type of protein. Urine contains various amounts of bicarbonate (up to about 200 mM). If high levels of bicarbonate are present in the urine sample, dilute the bicarbonate or pretreat the sample (eg, by molecular size exclusion)
Otherwise, sufficient bicarbonate would be brought into the assay to produce a very alkaline state, which would cause the dye-metal ion-protein interaction to have a low pH. Bottom (1.5-3.5)
And / or bicarbonate consistently interferes with the coordination of metal ions thereon, making the assay unreliable and useless. It is desired to provide a dry analytical element for protein measurement as follows. That is, a signal measurement that is substantially unaffected by the presence of a reducing agent or bicarbonate and that substantially correlates with the mass of protein present, ie, substantially ("substantially", refers to the total Means appropriate or acceptable to measure a particular protein, such as a measure of protein)
Generate signal measurements independent of protein type,
There is a need for a dry analytical element that can be used to quantitate proteins in the range of about 5 to about 300 mg / dL and that does not require pre-dilution, pre-concentration or pre-treatment of the sample to remove the interfering substances.

Unexpectedly, dry analytical elements suitable for use in measuring the amount or presence of proteins in biological fluids, ie, polymers containing acrylamide in the presence of molybdate ions And a dry analytical element containing an indicator dye together with a hydroxycarboxylic acid compound.

[0015]

SUMMARY OF THE INVENTION The problem with the prior art dry analytical element used to measure protein concentration is that the dry analytical element of the present invention, in the presence of molybdate ions,
It was overcome by using an indicator dye combining an acrylamide polymer and a hydroxycarboxylic acid compound. Molybdate is a preferred metal ion for use in embodiments of the present invention. However, embodiments of the present invention are not limited to molybdate ions only. Other suitable metal ions, such as tungstate ions, are also considered to be within the scope of the present invention.

As noted above, it is very important that different proteins react equivalently with the dye to produce a light signal that is related to the mass of the protein present in the sample and independent of the type of protein. Desirable. Unexpectedly, it was discovered that the polymeric media used to coat the various reagents of the dry analytical element of the present invention affected the apparent reactivity of the indicator dye system with different proteins. It was done. Thus, the intensity of the measured light signal is dependent on the type or type of protein present, but this dependence on the type of protein is significant when a polymer containing acrylamide is present in the dry analytical element. , Significantly lower.

A dry analytical element used for quantifying total protein in a sample, which measures the change in reflection density at an appropriate wavelength when a complex of an indicator dye and a protein is formed in the presence of ammonium molybdate. A dry analytical element based on this is disclosed herein. The dry elements of the present invention are useful for measuring the amount of protein in any liquid sample, especially biological fluids.

More specifically, in one embodiment,
The present invention provides a dry analytical element useful for measuring and quantifying proteins, comprising: (i) a porous developing layer; and (ii) one or more additional developing layers in fluid contact with the porous developing layer. And (iii) a support; wherein the element reacts upon contacting the element with at least one of the layers with a fluid suspected of containing protein. Producing a measurable change in the spectral absorption or reflection density of the dye,
An acrylamide-containing polymer, an indicator dye and a molybdate ion, and wherein the dye, the molybdate and the acrylamide-containing polymer are present together in the same layer, or optionally in separate layers of the element. Or dry analytical elements present individually.

The present invention provides the following method using the above-mentioned dry analytical element for measuring and quantifying a protein, and the method comprises the following method (A):

[0020]

Embedded image

(Wherein R ¹ and R ² are independently H, -CH
₃ is a -CH ₂ CH ₃ or -CH ₂ OH, and M is expected to hydroxycarboxylic acid represented by H or positively charged metal or counterion non-metallic), it contains a protein (B)
The fluid sample containing the hydroxycarboxylic acid,
Contacting with the analytical element; and (C) measuring the change in the spectral absorption or reflection density of the dye as a measure of the presence and amount of protein.

In a preferred embodiment, the present invention relates to the following analytical elements useful for measuring and quantifying proteins, the analytical elements comprising: (i) a porous developing layer; and (ii) a porous developing layer. Said element comprising one or more additional layers in fluid contact; and (iii) a support; wherein at least one of these layers comprises a polymer containing acrylamide, and The element has the formula:

[0023]

Embedded image

(Wherein R ¹ and R ² are independently H, -CH
₃ is a -CH ₂ CH ₃ or -CH ₂ OH, and M contains a hydroxycarboxylic acid represented by H or positively charged metal or counterion nonmetal), and further, the element Containing an indicator dye and molybdate ions that react upon contact with a fluid suspected of containing protein, causing a measurable change in the spectral absorption or reflection density of the dye, and the dye, The molybdate, the acrylamide-containing polymer and the hydroxycarboxylic acid are together present in the same layer, or are optionally combined or individually present in separate layers of the element; the analytical element.

The present invention provides the following method using the above-mentioned preferable dry analytical element for performing measurement and quantification of a protein, and the method is expected to contain (A) a protein. Contacting a fluid sample with said analytical element; (B) as a measure of the presence and amount of protein
Measuring changes in spectral absorption or reflection density.

[0026]

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the determination of proteins in a variety of aqueous fluids, such as human and animal biological fluids, food, industrial or municipal sewage, and other fluids commonly tested in the above-described manner. It is advantageously used to determine the presence and / or concentration. Biological fluids that can be tested include, but are not limited to, whole blood, serum, plasma, urine, cerebrospinal fluid, tear fluid, synovial fluid, lymph, tissue or platelet suspension, gingival fluid, vaginal fluid, cerebral fluid And other fluids readily apparent to those skilled in the art.

The proteins that can be measured include, but are not limited to, peptides, polypeptides, proteins (eg, enzymes, antibodies, lipoproteins and glycoproteins), and peptides, polypeptides and / or Or compounds that contain proteins or are linked (covalently or non-covalently) to these. The present invention is particularly useful for measuring protein in urine.

The present invention relates to a porous spreading layer, usually a diluted or undiluted test sample (eg, 1 to 5).
0 μL) is carried out using an analytical element comprising a coat layer having a porosity suitable for containing the same. The elements of the present invention are assembled using methods known in the art. Preferably, the porous spreading layer is isotropically porous, the properties of which are provided by interconnecting the spaces between the particles, fibers or other physical elements of the layer. The term "isotropically porous" means that the fluid is spread evenly throughout the layer. Useful materials for such layers are insoluble in water and maintain their structural integrity during the assay. Conventional materials and means used to assemble analytical elements are described, for example, in Przybylowicz et al., US Pat.
U.S. Pat. No. 4,258,00 to Pierce et al.
No. 1, Kitazima et al., U.S. Pat. No. 4,292,272 and Koyama et al., U.S. Pat. No. 4,430,436. The entire contents of these documents are incorporated herein by reference. A preferred porous spreading layer is Przybylowi
It is prepared by incorporating barium sulfate in ESTANE as described in US Pat. No. 3,992,158 to Ccz et al.

The analytical element of the present invention has one or more additional layers, all of which are in fluid contact with the porous spreading layer. The term "fluid contact" as used herein means that fluid can easily move from one layer to another. Such additional layers, preferably coated polymer layers, include an underlayer that blocks reagents and radiation, and one or more of the art-known ones such as gelatin and vinylpyrrolidone polymers. It is composed of a hydrophilic binder. Some layers are water-insoluble and others may be water-soluble.

The layers of the element of the present invention may be self-supporting, but preferably they are disposed on a dimensionally stable, chemically inert support. The support is
Preferably it is non-porous and transmits electromagnetic radiation. Preferred supports must be compatible with the specified mode of detection (eg, transmission or reflectance spectroscopy). Useful support materials include, but are not limited to, paper, metal foils, polystyrenes, polyesters, polycarbonates and cellulose esters.

In at least one of the layers of the element of the present invention, a dye capable of reacting specifically with proteins and molybdate ions is present. The term “dye that reacts with protein and molybdate” as used herein refers to the spectral or optical absorption properties of the dye in the presence of molybdate and protein, when the protein is not present. Any dye compound that differs from the above properties of the dye and molybdate ion is meant. Dyes having the desired properties described above include an open aromatic structure having a functional group capable of coordinating molybdate ions (eg, but not limited to, two adjacent hydroxyl groups on an aromatic ring). st
ructure) and dyes comprising a tricyclic aromatic structure. Such dyes include, but are not limited to, pyrocatechol violet, tetrabromophenolphthalein ethyl ester, triiodophenolsulfonephthalein, tetrabromopyrogallol red and pyrogallol red.

In a preferred embodiment of the present invention, there is provided a multi-layer analytical element for use in determining the presence and / or amount of a protein. Specifically, the multi-layer element comprises a non-porous support upon which, in fluid contact,
(I) a first reagent or buffer layer containing a dye that reacts with proteins and molybdate ions, molybdate, an acrylamide-containing polymer, (ii) a lower layer, and (ii)
i) a porous development layer.

The element of the present invention may contain, in suitable layers, various additives known in the art to assist in manufacturing, spreading the fluid, and absorbing unwanted radiation.
The elements of the present invention can be manufactured using conventional coating methods and equipment described in the prior art, including coating methods such as gravure, curtain, and hopper methods. The elements of the present invention can be arranged in various forms, including elongated tapes, sheets, slides or chips of any desired width. Further, the methods of the present invention can be performed manually or automatically using appropriate analyzers and methods. In general, the method of the present invention is carried out by spotting a test sample (for example, 1 to 50 μl) on a porous spreading layer, so as to come into contact with the reagent of the analysis element. The fluid moves through the analytical element, effectively mixing the reagents and the reaction takes place.

After application of the sample, the analytical element accelerates the formation of the protein-dye-molybdate complex.
Alternatively, it is exposed to any conditioning, such as incubation, heating, etc., that may be desirable to facilitate. As noted above, dyes utilized in the present invention that react with proteins and molybdate ions include, but are not limited to, functional groups capable of coordinating molybdate ions, such as, but not limited to, adjacent aromatic rings. There are dyes comprising an open aromatic structure having two hydroxyl groups) and a tricyclic aromatic structure. Such dyes include, but are not limited to, pyrocatechol violet, tetrabromophenolphthalein ethyl ester, triiodophenolsulfonephthalein, tetrabromopyrogallol red and pyrogallol red.

The above dye compound is a known chemical maker,
For example, Eastman Organic Chemical Company, Aldrich Ch
It can be purchased from the emical Company, Sigma Chemical Company, or the like, or can be prepared using conventional starting materials and methods known to those skilled in the art. Dry analytical elements having the following indicator dyes separately were prepared. The pigment is pyrocatechol violet [pyrocatechol, 4,4 '-(3H-2,1-benzoxathiol-3-ylidene) -di-S, S-dioxide]; pyrogallol red [2- (4,5, 6-trihydroxy-
3-oxo-3H-xanthen-9-yl) benzenesulfonic acid]; bromopyrogallol red [spiro (3H
-2,1-benzoxathiol-3 ', 9'-[9H]
-Xanthene-3 ', 4', 5 ', 6'-tetrol-
2,7-dibromo-I, I-dioxide)]; galein [3 ', 4', 5 ', 6'-tetrahydroxyspiro [isobenzofuran-1 (3H), 9'-[9H] xanthene] -3 -On]. General characteristics of dry analytical elements are described in US Pat. No. 3,992,158 to Przybylowicz et al.
No. 4,258,00 to Frank and Pierce.
No. 1. All of these dyes are sensitive to changes in the physicochemical environment.

We have discovered that urine protein can be quantitatively analyzed using the above dye in combination with molybdate ions in a dry analytical element. The dry analytical element comprising pyrocatechol violet in combination with molybdate ions contains the highest analytical detection sensitivity (ie the highest change in reflection concentration Dr over the desired protein concentration range up to 300 mg / dL) and contains human albumin Repeat precision was provided for the prepared solutions.

We have unexpectedly found that the polymeric medium used to coat the various reagents of the dry element affects the apparent reactivity of the indicator dye system with different proteins, Thus, it has been found that the intensity of the measured light signal affects the dependence on the type of protein present. Polymers suitable for use in the present invention have the effect of flattening a light signal,
That is, the polymer has the effect of reducing the difference between the measured values of the reflection density signal obtained for proteins of equal mass or different weight. This is desirable because different types of proteins predominate in different samples, and the desired measurement is the total mass of protein present per unit volume of sample (total protein). Ideally, a total protein assay should quantify all proteins with equal sensitivity. Examples of the polymer of the present invention include, but are not limited to, a water-soluble homopolymer,
There are copolymers and terpolymers, mostly composed of acrylamide (greater than about 40% by weight). The functional groups capable of coordinating molybdate ions, when present in the polymers of the present invention, must not be present in amounts that would interfere with protein measurements. Preferred polymers are homopolymers of acrylamide, i.e., having a weight average molecular weight of about 20,000-25.
It is a polyacrylamide of 000 dalton (hereinafter referred to as poly A). The most preferred range is about 100,0
00-150,000 daltons.

In addition, we have quite unexpectedly discovered that when glycolic acid is added to a multi-layered analytical element (preferably pre-coating the analytical element), bicarbonate causes He found that the disturbance was substantially reduced. General structural formula:

[0039]

Embedded image

(Wherein R ¹ and R ² are independently H, -CH
₃ is a -CH ₂ CH ₃ or -CH ₂ OH, and M is other hydroxycarboxylic acids represented by H or positively charged metal or counterion nonmetal) is have similar effects Therefore, it is suitable for use in the present invention. Representative hydroxycarboxylic acids represented by the above formula include 2-hydroxypropanoic acid and 2-methyl-2
-Hydroxypropanoic acid and their salts.

Satisfactory quantitative analysis of urine protein can be obtained using a dry analytical element comprising the indicator dye and molybdate ion in combination with the acrylamide polymer and hydroxycarboxylic acids. Pyrocatechol violet and molybdate ions combined with a polyacrylamide polymer having a molecular weight of about 20,000 to 250,000 daltons and also glycolic acid are preferred dry analytical elements, with a sensitive measure of protein concentration up to 300 mg / dL and very high Provides excellent repeatability. The most preferred dry analytical element contains polyA (average molecular weight of 100,000 to 150,000 daltons) as a coated polymeric medium and a precoated glycolic acid in a reagent / buffer layer.

Polyacrylamide has an action of flattening the reactivity of proteins different from the indicator dye pyrocatechol violet / molybdate ion system.
The difference in reflection density signal for different types of proteins of the same weight concentration is significantly reduced in dry elements using the preferred polymer. The effect of polymers using pyrocatechol violet and molybdate is observed when using other types of indicator dyes and metal ions. The present invention can produce dry elements used to quantify total protein in urine in the range of about 5-300 mg / dL. The dry elements of the present invention are nearly equivalent in sensitivity to different proteins, so that they are suitable for their intended use and provide greater accuracy than prior art solution and dry element based assays. The main interfering effect due to the wide range of bicarbonate concentrations present in urine samples is due to the addition of glycolic acid to the dry element, which utilizes the pyrocatechol-molybdate ion chemistry. Was overcome. Other hydroxycarboxylic acids, such as those disclosed above, have a similar effect whether incorporated into a dry element or added to a sample. These two very different and unexpected findings provide a sensitive and powerful dry assay element that is particularly suitable for quantifying total protein in liquid samples.

[0043]

The scope of the present invention will be described below with reference to examples. These embodiments are shown for illustrative purposes only, and the invention specifically described in the embodiments should not be limited to the embodiments. Unless otherwise noted, all reagents and equipment were obtained from the manufacturer.

The following Examples 1 to 4 show the results of experiments comparing the sensitivity and repetition accuracy of analytical detection of analytical elements containing different indicator dyes. EXAMPLE 1 pyrocatechol violet pyrocatechol violet, in an amount of 0.12 g / m ^2, on the outer, the following composition of 12 g / m ² [poly (acrylamide - co -N- vinyl-2-pyrrolidone), 50 :
Acrylamide polymer (hereinafter, referred to as 50 weight ratio)
Polymer AVP), Zonyl FS surfactant
N (0.36 g / m ² ) and succinic acid as buffer 5.
9 g / m ² (pH 2.5), ammonium molybdate (0.18 g / m ² ) and potassium oxalate (0.1
5 g / m ² ) and coated in a reagent layer containing 5 g / m ² ).
A bead spreading layer (having the following composition described in US Pat. No. 4,258,001) was used. A lower layer of poly (N-vinyl-2-pyrrolidone) was provided between the developing layer and the reagent layer. The element was cut into 1 cm ² squares and mounted on slides. Johnson & Johnson Clinical Diagnosti
c Using a slide analyzer, a protein-containing solution was spotted on the slide elements, the slides were incubated, and the reflection density Dr was read. According to this method of evaluating components, a measurer can test a large number of components and easily obtain repeated measurements. Other methods of spotting, incubating and then measuring the light signal are also suitable for evaluating dry elements. The structure of the element is shown below. Elemental development layer of pyrocatechol violet Copoly (vinyltoluenemethacrylic acid) 30 μm beads Lower layer poly (N-vinyl-2-pyrrolidone) copoly (acrylamide-co-N-vinyl-2-pyrrolidone) (50:50 weight ratio) Pyrocatechol Violet Reagent Layer Ammonium Succinate Molybdate Carboxyl Oxalate Zonyl FSN ™ Support Dr was measured at 670 nm. Table 1 below shows the test results obtained using the pyrocatechol element described above. The fluid containing the protein contains a known weight of human serum albumin,
It was prepared by adding a known volume of a 0.15 M aqueous sodium chloride solution to obtain a fluid having a concentration of albumin shown in Table 1. The fluid 1 containing albumin
Each 0 μL was spotted on the development layer of the analysis element. The spotted elements were incubated at 37 ° C. for 5 minutes. Next, the reflection density was measured. The measured value of Dr is the average value (<Dr>) of six repeated measurements (n-6). % CV is a coefficient of variation of the Dr average value.

Table 1 HSA (mg / dL) <Dr>% CV 10 1.022 4.8 50 1.209 1.0 100 1.370 2.3 150 1.489 1.9 300 1.732. 7 Change in reflection density of 10 to 300 mg / dL albumin (ΔD
(r = 0.710), the detection sensitivity of the pyrocatechol element is very excellent. Its% CV is relatively small, indicating excellent repeatability. Example 2 Pyrogallol Red Pyrogallol Red was coated in an amount of 0.18 g / m ^{2 in} a barium sulfate spreading layer described in US Pat. No. 3,992,158. Surfactant TR
ITON X-10 (0.001 g / m ² ), tartaric acid (6.0 g / m ² , pH 2.5) and oxalic acid (2 g / m ² )
m ² ) was changed to a acrylamide homopolymer poly A (average molecular weight 100,000 daltons) with a coverage of 10 g / m 2
Coated in the reagent layer used in ² . Ammonium molybdate was applied in an amount of 0.9 g / m ^{2 in} a barium sulfate spreading layer. A lower layer of poly (N-isopropylacrylamide) was coated between the spreading layer and the reagent layer. The structure of the element is shown below. Estar support coated with several buffers at pH 2.5, but tartaric acid showed the most linear response (at 540 nm) over the concentration range of proteins tested. The experimental protocol is
Same as described for the pyrocatechol violet element, except that Dr was measured at 540 nm. Table 2 shows the test results (n = 6).

Table 2 HSA (mg / dL) <Dr>% CV 100 0.281 5.0 50 0.327 4.9 150 0.363 7.1 300 0.382 4.4 Using Pyrogallol Red The change in Dr over the range of albumin concentrations tested was 0.101,
The detection sensitivity is lower than that obtained with the pyrocatechol violet element. The% CV is small but somewhat larger than measured using the pyrocatechol violet component. Elements Bromopyrogallol Red in Example 3 Bromopyrogallol Red in an amount of 0.24 g / m ^2, poly polyacrylamide medium 12 g / m ² (acrylamide - co-N-(3- acetoacetamidopropyl) methacrylamide) (95: 5 weight ratio) (hereinafter referred to as polymer AAM). Surfactant TRITON X-165 (0.20 g
/ M ² ), malonic acid (8.0 g / m ² , pH 2.5) and ammonium molybdate (0.4 g / m ² ) are also AA
It was coated in the M layer. A barium sulfate spreading layer was coated over a poly (N-isopropylacrylamide) underlayer. The structure of the element containing barium sulfate is shown below. Bromopyrogallol red element developing layer Barium sulfate lower layer poly (N-isopropylacrylamide) poly (acrylamide-co-N- (3-acetoacetamidopropyl) methacrylamide) (95: 5 weight ratio) Bromopyrogallol red Reagent layer Malonic acid Ammonium molybdate Potassium oxalate bisvinylsulfonylmethyl ester (BVSME) Estar-based bromopyrogallol elements were evaluated as described above for elements containing pyrocatechol and pyrogallol red indicator dyes. Bromopyrogallol (n =
6) Table 3 shows data obtained by evaluating the elements.

Table 3 HSA (mg / dL) <Dr>% CV 10 1.482 86.1 50 1.487 20.6 100 1.547 11.3 300 1.660 2.6 Through concentration range of albumin The change in Dr is 0.17
8, the detection sensitivity is lower than that of pyrocatechol violet. % CV is large. This indicates that the repeat accuracy is significantly lower. EXAMPLE 4 Gullane Gullane, in an amount of 0.12 g / m ^2, was coated placed in barium sulfate spreading layer. Surfactant TRITON
X-165 (0.2 g / m ² ), malonic acid (8.0 g / m ² )
m ² , pH 2.5) and oxalic acid (2.5 g / m ² )
Having a molecular composition of poly (acrylamide-co-N-vinyl-2-pyrrolidone) (50:50 weight ratio), hereinafter referred to as polymer AVP (10.0 g / m ² )
To form a buffer layer. Ammonium molybdate (0.90 g / m ² ) and surfactant TRI
TON X-100 (1 g / m ² ) was added to the dye and coated in a barium sulfate spreading layer. The structure of the element is shown below. Element of galein Barium sulfate spread layer Galein ammonium molybdate TRITON X-100 (trademark) lower layer poly (N-isopropylacrylamide) poly (acrylamide-co-N-vinyl-2-pyrrolidone) (50:50 weight ratio) buffer layer malon Acid TRITON X-165 Estar oxalate support The gallein elements were evaluated as described above for the other elements (n = 6), and the Dr was measured at 550 nm. Table 4 shows the test results.

Table 4 HSA (mg / dL) <Dr>% CV 10 0.541 85.7 50 0.569 20.3 100 0.565 93.1 300 0.580 19.5 Through concentration range of albumin The change in Dr is 0.03
9, the detection sensitivity is considerably lower than that of pyrocatechol violet. % CV is quite large,
This indicates that the repeatability is quite low.

In this example, pyrocatechol violet
The preferred dye and ammonium molybdate
Different types of tans using dry analytical elements
Effect of polymer media with different reagent layers on protein measurement
The fruit was tested. The polymer compared in this experiment was poly A
(Weight average molecular weight is about 120,000 daltons);
Li (acrylamide-co-N-vinyl-2-pyrrolide
), 50/50 weight ratio] (polymer AVP);
Composition (poly (polyacrylamide-co-acryl)
Acid), a polymer having a 90/10 weight ratio)
(Referred to as merged PAA). The deployment layer is in all cases,
No. 3,992,156.
Thus, barium sulfate was used. The polymers are dried
Coverage (12g / m ^Two). Contains protein
The solution is a known weight of the purified human protein of interest (I
gG, retinol binding protein and kappa light chain
) With a known volume of a 0.15 M aqueous sodium chloride solution
Prepared by adding The factors are as described above.
Slides. Calibrate the assay with purified human
Use a solution containing albumin (hereinafter referred to as calibration fluid)
I went there. Contains 100mg / dL test protein
Separate dry analysis (slide) for 10 μL of each solution
They were spotted individually. Slide the slides at 37 ° C for 5 minutes
After incubation, measure the reflection density at 670 nm.
Was. Measure the level of total protein in the sample
Calculated from the calibration calibration method using the
Was. Table 5 shows the results.

TABLE 5 Effect of polymer type on protein reactivity Polymer albumin κ light chain λ light chain IgG retinol-linked α-1-miku mg / dL mg / dL mg / dL mg / dL combined protein globulin mg / DL mg / dL AVP 100 33 33 64 62 44 Poly A 100 44 32 73 73 66 66 PAA 100 ND ^** 34 59 NDND ^** Not measured The data in Table 5 shows that the polymer was composed of pyrocatechol and molybdate ion. It has been shown to affect protein interactions. Since polyA has in particular the effect of reducing the difference in Dr measurements between protein types, the estimated protein concentration is determined according to a calibration method using a fluid containing human albumin. As a result, the expected 10% for the amount of protein added to the fluid
An estimate of the protein concentration approaching 0 mg / dL was obtained. Poly A had the highest function, ie, the least dependence of the measured signal on the type of test protein. Example 6 In this example, the effect of glycolic acid on interference by bicarbonate was evaluated. Glycolic acid converts poly A to 12
It was precoated in a reagent layer containing g / m ² . Glycolic acid was coated in the reagent layer at the level shown in Table 6. Sodium bicarbonate was added to the pooled human urine sample to bring the final bicarbonate concentration to 100 mM or 200 mM.
(In both cases, the pH of the final pooled urine sample was 8.5). The pH was not adjusted to the final measurement, but was still after addition of bicarbonate.
Total protein in untreated urine pool control sample is 18 mg / dL
(Measured using the BIOTROL® kit for total protein determination, which is based on a solution-based assay using the dye-binding method of pyrogallol red / ammonium molybdate, and is based in Pennsylvania, USA 19341, Biotro, Ixton
l Obtained from Diagnostics. Catalog number: A01
217U). The reflection concentration was measured in the same manner as in Example 5 above, and the total protein concentration was determined using the Dr value measured after calibrating the test element using the same fluid containing human albumin as the calibration fluid as in Example 5. Was estimated. The protein estimates obtained for this control urine were subtracted from the protein estimates obtained for bicarbonate-treated urine samples. The data is shown in Table 6.

[0051]Table 6 Effect of glycolic acid on bicarbonate interference. Differences in protein concentration between bicarbonate-treated urine and control urine without bicarbonate Glycolic acid Bicarbonate g / m ²  100mM 200mM 0 35 110 0.125 22 77 0.25 13 54 0.375 8 30 0.5 420 20 0.75 N.P. D. 131.0 N.I. D. -2 (ND = not measured) Bicarbonate produces a light signal similar to protein
There is action. For example, without glycolic acid, bicarbonate
The estimated protein concentration of a urine sample with a salt concentration of 200 mM is
About 110 mg / dL higher than the current level. As shown in Table 6
Glycolic acid dramatically reduces the effects of bicarbonate
Let it. Glycolic acid (or other suitable
Hydroxycarboxylic acids) are dry fractions as in Example 6.
May be added directly to the analytical element, or
May be added to the sample before contacting the sample. Analysis required
A useful concentration range for glycolic acid coated on
0.125 to 2.0 g / m^TwoIt is. Its preferred range
Is 0.125 to 1.5 g / m ^TwoAnd the more preferred range
The range is 0.25-1.25 g / m^TwoIt is. Preferred dry type
The structure of the analysis element is shown below. Deployment layerBarium sulfate Underlayer Poly (N-isopropylacrylamide) Polyacrylamide (Poly A) Glycolic acid Pyrocatechol violet dye Reagent / buffer layer Ammonium molybdate Potassium oxalate Zonly-FSN surfactantMalonic acid buffer pH = 2.5 The above experiments and examples are intended to illustrate the scope and spirit of the present invention.
It is provided for the purpose. These embodiments and examples
For those skilled in the art, other embodiments and implementations are possible.
Examples will become apparent. These other embodiments and implementations
Examples are within the spirit of the invention. Therefore, the present invention
Should be limited only by the claims herein.
It is.

 ──────────────────────────────────────────────────の Continuation of front page (72) Inventor David B. Ratato United States, New York 14617, Rochester, Cinnabar Road 285 (72) Inventor John C. Mauk United States, New York 14610, Rochester, Croydon Road 184 (72) Inventor James Earl. Scafer USA, New York 14526, Penfield, Jackson EK Stee. Lord 49 (72) Richard Sea. Ston United States of America, New York 14617-5216, Rochester, Twilight Drive 24 (72) Inventor Wayne W.W. Webber, The Second United States, New York 14472, Mendon, Partridge Hill Road 38 (72) Robert F. Inventor. Winter Cone United States, New York 14609, Rochester, Westchester Avenue 330

Claims (36)

[Claims] 1. A dry analytical element for measuring a protein, comprising: (A) a porous developing layer; (B) (i) reacting with a protein and molybdate ions to change its own spectral absorption or reflection density. (Ii) a molybdate, and (iii) a polymer containing acrylamide, wherein the dye, the molybdate and the polymer containing acrylamide are both present in the same layer. A dry analytical element comprising: (C) a support; or one or more additional layers, either in any combination or individually in separate layers of said element. 2. The dry analytical element according to claim 1, wherein the dye is selected from the group consisting of pyrocatechol violet, pyrogallol red, bromopyrogallol red and galein. 3. The dry analytical element according to claim 2, wherein the dye is pyrocatechol violet. 4. The dry analytical element according to claim 1, wherein the molybdate is ammonium molybdate. 5. The polymer containing acrylamide is poly A, polymer AVP, polymer PAA and polymer AAM.
The dry analytical element according to claim 1, which is selected from the group consisting of: 6. The polymer according to claim 1, wherein said polymer has a molecular weight range of 100,
The dry analytical element according to claim 5, which is poly A of 000 to 150,000 daltons. 7. The polymer, wherein the dye is pyrocatechol violet, the molybdate is ammonium molybdate, and the acrylamide-containing polymer is
The dry analytical element according to claim 1, which is poly A having a molecular weight range of 100,000 to 150,000 daltons. 8. The dry analytical element according to claim 7, wherein the porous developing layer contains barium sulfate. 9. A method for measuring a protein, comprising: (A) a fluid expected to contain a protein,
General structural formula: (Wherein R ¹ and R ² are independently H, —CH ₃ , —CH ₂
CH ₃ or —CH ₂ OH, and M is H or a positively charged metal or non-metal counter ion)
(B) mixing the fluid containing the hydroxycarboxylic acid and expected to contain a protein, (a) a porous developing layer, (b) (i) A dye capable of changing its spectral absorption or reflection density by reacting with protein and molybdate ions, (ii) molybdate, (iii) a polymer containing acrylamide, One or more additional layers in which the molybdate and the polymer comprising the acrylamide are both present in the same layer, or in any combination or individually, in separate layers of the element. Contacting a dry analytical element comprising: a layer in fluid contact with the porous spreading layer; (c) a support; and (C) a measure of protein. And measuring the change in spectral absorption or reflection density of the dye. 10. The method of claim 9, wherein said dye is selected from the group consisting of pyrocatechol violet, pyrogallol red, bromopyrogallol red, and galein. 11. The method according to claim 10, wherein said dye is pyrocatechol violet. 12. The method of claim 9, wherein said molybdate is ammonium molybdate. 13. The polymer containing acrylamide,
Poly A, polymer AVP, polymer PAA and polymer AA
10. The method of claim 9, wherein the method is selected from the group consisting of M. 14. The polymer having a molecular weight range of 10
0000-150,000 dalton poly A,
The method according to claim 13. 15. The method of claim 15, wherein the dye is pyrocatechol violet, the molybdate is ammonium molybdate, and the acrylamide polymer is poly A having a molecular weight range of 100,000 to 150,000 daltons. Item 10. The method according to Item 9. 16. The method of claim 15, wherein said porous spreading layer comprises barium sulfate. 17. A dry analytical element for measuring protein, comprising: (A) a porous developing layer; (B) (i) reacting with a protein and molybdate ions to change its own spectral absorption or reflection density. (Ii) molybdate, (iii) a polymer containing acrylamide, (iv) a general structural formula: (Wherein R ¹ and R ² are independently H, —CH ₃ , —CH ₂
CH ₃ or —CH ₂ OH, and M is H or a positively charged metal or non-metal counter ion)
Wherein the dye, the molybdate, the polymer comprising acrylamide and the hydroxycarboxylic acid are both present in the same layer, or separate from the element. One or more additional layers in any combination or individually in a layer, the layer being in fluid contact with the porous spreading layer, and (C) a support. Dry analytical element. 18. The method according to claim 17, wherein the dye is selected from the group consisting of pyrocatechol violet, pyrogallol red, bromopyrogallol red and galein.
The described dry analytical element. 19. The dry analytical element according to claim 18, wherein the dye is pyrocatechol violet. 20. The dry analytical element according to claim 17, wherein said molybdate is ammonium molybdate. 21. The polymer containing acrylamide,
Poly A, polymer AVP, polymer PAA and polymer AA
18. The dry analytical element according to claim 17, selected from the group consisting of M. 22. The polymer according to claim 1, wherein the polymer has a molecular weight range of 100,
22. The dry analytical element according to claim 21, which is 000 to 150,000 dalton poly A. 23. The method of claim 23, wherein the hydroxycarboxylic acid is glycolic acid, 2-hydroxypropanoic acid, 2-methyl-2-
18. The dry analytical element according to claim 17, selected from the group consisting of hydroxypropanoic acid and salts thereof. 24. The dry analytical element according to claim 23, wherein said hydroxycarboxylic acid is glycolic acid. 25. The method of claim 25, wherein the pigment is pyrocatechol violet, the molybdate is ammonium molybdate, the acrylamide polymer is poly A having a molecular weight range of 100,000 to 150,000 daltons, and The dry analytical element according to claim 17, wherein the hydroxycarboxylic acid is glycolic acid. 26. The dry analytical element according to claim 25, wherein the porous spreading layer contains barium sulfate. 27. A method for measuring a protein, comprising: (A) a fluid sample expected to contain a protein; (a) a porous developing layer; (b) (i) a protein and molybdate ions. (Ii) molybdate, (iii) polymer containing acrylamide, (iv) general structural formula: (Wherein R ¹ and R ² are independently H, —CH ₃ , —CH ₂
CH ₃ or —CH ₂ OH, and M is H or a positively charged metal or non-metal counter ion)
Wherein the dye, the molybdate, the polymer comprising acrylamide and the hydroxycarboxylic acid are both present in the same layer, or separate from the element. Present in any combination or individually in a layer,
One or more additional layers in fluid contact with the porous spreading layer; (c) a support;
And (B) measuring the change in spectral absorption or reflection density of the dye as a measure of protein. 28. The dye of claim 27, wherein the dye is selected from the group consisting of pyrocatechol violet, pyrogallol red, bromopyrogallol red, and galein.
The described method. 29. The method of claim 28, wherein said dye is pyrocatechol violet. 30. The method of claim 27, wherein said molybdate is ammonium molybdate. 31. The polymer containing acrylamide,
Poly A, polymer AVP, polymer PAA and polymer AA
28. The method of claim 27, wherein the method is selected from the group consisting of M. 32. The polymer containing acrylamide,
32. The method of claim 31, wherein the molecular weight is poly A having a molecular weight range of 100,000 to 150,000 daltons. 33. The hydroxycarboxylic acid is glycolic acid, 2-hydroxypropanoic acid, 2-methyl-2-
28. The method of claim 27, wherein the method is selected from the group consisting of hydroxypropanoic acid and salts thereof. 34. The method according to claim 33, wherein said hydroxycarboxylic acid is glycolic acid. 35. The pigment is pyrocatechol violet, the molybdate is ammonium molybdate, the acrylamide polymer is poly A having a molecular weight range of 100,000 to 150,000 daltons, and the hydroxy 28. The method according to claim 27, wherein the carboxylic acid is glycolic acid. 36. The method of claim 35, wherein said porous spreading layer comprises barium sulfate.